The objectives of this project are to: (1) investigate the interaction of alcohol with proteins and lipids in biological membranes; (2) study structure and dynamics of membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3; and (3) study lipid-protein interactions related to alcoholism and lipid polyunsaturation. (1) We obtained direct evidence by NMR that ethanol interacts preferentially with the lipid-water interface of membranes. Ethanols interactions are driven by both the opportunity for hydrogen bonding and hydrophobic interactions. We quantitated ethanol binding to membranes composed of lipids and proteins at the physiological ethanol concentration of 20 mM by headspace gas chromatography. This method is ideally suited for partitioning studies because it is non-perturbing. Under physiological conditions, of the order of 10% of total ethanol is bound to the interfaces of lipids and proteins. NMR measurements indicate that free and bound ethanol molecules are in rapid exchange, and that ethanol passes through membranes at rates that are only slightly lower than permeation rates of water. Interfacial binding of ethanol raises effective ethanol concentrations at surfaces but lowers its concentration in the electrolyte solutions of living organisms. The interface location of ethanol lowers interfacial energy of lipids and proteins. In lipid membranes this results in an increase of area per lipid molecule and a disordering of lipid hydrocarbon chains. Ethanol-induced chain disordering is smaller in polyunsaturated bilayers, most likely because polyunsaturated hydrocarbon chains already occupy a larger area per molecule and are therefore less sensitive to ethanol-induced disordering. The ethanol molecules at the lipid-water interface block pathways for water diffusion through lipid bilayers as seen in decreased rates of water permeation. (2) The membranes of brain synaptosomes and retinal rod outer segments contain 30-50 mol% of the six-fold unsaturated docosahexaenoic acid (DHA) as lipid hydrocarbon chains. One possible role of DHA is to alter membrane mechanical properties important for activity of receptor proteins. There is controversy as to the nature of the perturbation which DHA chains induce on membrane hydrocarbon order. The six methylene-interrupted cis double bonds within DHAs 22 carbon unit reduce the number of degrees of freedom for structural transitions, which led to the suggestion that these chains have a specific rigid conformation such as angle-iron or helical. However, direct measurements of DHA chain order parameters reveal a different picture. Using a magic angle spinning NMR experiment which re-couples 13C-1H dipolar interactions, assigned DHA order parameters were obtained, and dimensions of the DHA chain unit cell were determined by x-ray diffraction. The results suggest that DHA chains in membranes prefer looped conformations and undergo rapid structural transitions, providing increased flexibility to receptor-rich neural membranes. We developed quantitative methods for interpretation of NMR NOESY cross-relaxation rates between lipid resonances. In addition to providing information on lipid structure, these rates are sensitive to the dynamics of membrane reorganization in the correlation time range form pico- to microseconds. The comparison of experimental rates and rates from molecular dynamics calculations suggests that distance variation between protons caused by lateral diffusion of lipid molecules is the primary mechanism of cross-relaxation in lipids. The analysis quantifies the high degree of molecular disorder in biological membranes, showing a finite probability of close approach between even the most distant segments of neighboring lipid molecules (e.g. the methyl groups in the choline headgroup and the terminal methyl groups of the fatty acid chains). Intermolecular cross-relaxation rates are an ideal tool to study lateral lipid organization in the liquid-crystalline phase of lipids. Inhomogeneous lipid distribution and preferences in the interaction of lipid species, as well as preferences in the location of substances that incorporate into membranes can be detected. (3) The behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1 in membranes was investigated by solid state 2H NMR on P828 with the selectively deuterated isoleucines I3, I13, I16, and I20. The data are consistent with partial penetration of the N-terminal peptide region into the hydrophobic core of the membrane, while the C-terminal portion of the peptide remains near the lipid/water interface. Peptide incorporation results in a significant reduction of lipid chain order toward the bilayer center, but only a modest reduction near the lipid glycerol. In addition, the structure of the peptide was investigated free in water and bound to SDS micelles by high resolution NMR. P828 is unstructured in water but exists in a flexible, partially helical conformation when bound to negatively charged liposomes or micelles. The flexible helix covers the first 14 residues of the peptide, whereas the C-terminus of the peptide appears to be unstructured. The peptide-induced changes in lipid chain order profiles indicate that membrane curvature stress is the driving force for the cytolytic behavior of P828.